Abstract

Despite the ubiquitous presence of the COPI, COPII, and clathrin vesicle budding machineries in all eukaryotes, the organization of the secretory pathway in plants differs significantly from that in yeast and mammalian cells. Mobile Golgi stacks and the lack of both transitional endoplasmic reticulum (ER) and a distinct ER-to-Golgi intermediate compartment are the most prominent distinguishing morphological features of the early secretory pathway in plants. Although the formation of COPI vesicles at periphery of Golgi cisternae has been demonstrated in plants, exit from the ER has been difficult to visualize, and the spatial relationship of this event is now a matter of controversy. Using tobacco (Nicotiana tabacum) BY-2 cells, which represent a highly active secretory system, we have used two approaches to investigate the location and dynamics of COPII binding to the ER and the relationship of these ER exit sites (ERES) to the Golgi apparatus. On the one hand, we have identified endogenous COPII using affinity purified antisera generated against selected COPII-coat proteins (Sar1, Sec13, and Sec23); on the other hand, we have prepared a BY-2 cell line expressing Sec13:green fluorescent protein (GFP) to perform live cell imaging with red fluorescent protein-labeled ER or Golgi stacks. COPII binding to the ER in BY-2 cells is visualized as fluorescent punctate structures uniformly distributed over the surface of the ER, both after antibody staining as well as by Sec13:GFP expression. These structures are smaller and greatly outnumber the Golgi stacks. They are stationary, but have an extremely short half-life (<10 s). Without correlative imaging data on the export of membrane or lumenal ER cargo it was not possible to equate unequivocally these COPII binding loci with ERES. When a GDP-fixed Sar1 mutant is expressed, ER export is blocked and the visualization of COPII binding is perturbed. On the other hand, when secretion is inhibited by brefeldin A, COPII binding sites on the ER remain visible even after the Golgi apparatus has been lost. Live cell imaging in a confocal laser scanning microscope equipped with spinning disk optics allowed us to investigate the relationship between mobile Golgi stacks and COPII binding sites. As they move, Golgi stacks temporarily associated with COPII binding sites at their rims. Golgi stacks were visualized with their peripheries partially or fully occupied with COPII. In the latter case, Golgi stacks had the appearance of a COPII halo. Slow moving Golgi stacks tended to have more peripheral COPII than faster moving ones. However, some stationary Golgi stacks entirely lacking COPII were also observed. Our results indicate that, in a cell type with highly mobile Golgi stacks like tobacco BY-2, the Golgi apparatus is not continually linked to a single ERES. By contrast, Golgi stacks associate intermittently and sometimes concurrently with several ERES as they move.

Cross-Reactivities of Antisera Raised against Recombinant Arabidopsis COPII Proteins.(A) Cytosolic proteins and total membranes were isolated from the suspension cultures of Arabidopsis and tobacco BY-2 cells as described in Methods and probed with the antisera indicated. Equal amounts of protein were applied to the lanes in each gel blot (20 μg per lane for the Arabidopsis gel; 30 μg per lane for the BY-2 gel). M, membrane; C, cytosol.(B) Arabidopsis total cell membranes separated on a linear isopycnic sucrose density gradient (as described in Methods). Individual fractions were probed with COPII antisera and with standard antisera for ER (calnexin) and Golgi (reversibly glycosylated polypeptide [RGP]) marker proteins.

Expression of Sec13-GFP Is without Effect on Secretion.Tobacco protoplasts were electroporated with a constant amount of plasmid encoding for α-amylase together with increasing amounts of plasmid encoding for LeSec13-GFP or for the GTP-blocked mutant Sar1[H74L]. Standard deviations, as indicated by error bars, were calculated from six separate experiments. Protein gel blots of total cytosolic proteins, corresponding to the individual panels of the secretion index histogram, are also given to document the gradual increase in expressed effector protein in the protoplast homogenates. Note that in marked contrast with LeSec13-GFP, the expression of the Sar1 mutant leads to a drastic reduction in secretory activity.

Analysis of LeSec13:GFP Dynamic by FRAP.(A) to (D) FRAP analysis of LeSec13:GFP present within the nuclear envelope, ER, and cytosol. LeSec13:GFP fluorescence was monitored before (A), immediately after photobleaching (B), as well as 1 min (C) and 60 min (D) after recovery.(E) and (F) Detailed views of the boxed regions shown in (A) and (D), respectively.(G) to (J) FRAP analysis of nuclear LeSec13:GFP.(G) Plots show fluorescence recovery of nuclear LeSec13:GFP versus free GFP in the bleached area. Images were acquired every 30 s. Measurements of fluorescence intensity were subtracted from background fluorescence and normalized from loss of fluorescence during bleaching and imaging. Error bars are standard deviations (n = 3).(H) to (J) The images show single optical sections of LeSec13:GFP present within the nucleus before (H) and immediately after photobleaching (I) as well as 10 min after recovery (J). Bleached areas are circled in white.Bars = 5 μm.

Analysis of LeSec13:GFP Dynamic by Time-Lapse Microscopy.(A) to (D) Cortical LeSec13:GFP was visualized using an UltraVIEW RS confocal microscope. Single optical sections images were acquired every 1.2 s for 27.6 s (see Supplemental Video 1 online). Images taken at 0, 1.2, 13.2, and 27.6 s are presented ([A] to [D], respectively). Arrowheads and circles indicate LeSec13:GFP punctate structures that remained immobile for at least 1.2 and 13.2 s, respectively. Bars = 5 μm.(E) Number of fluorescent foci in each individual frames of the video were calculated using the analyze particles command in ImageJ and were plotted on a graph.

Effects of Sar1[T39N] Secretory Inhibitors on LeSec13:GFP.(A) to (F) LeSec13:GFP-expressing cells were transformed by biolistics so as to express SP:RFP alone ([A] to [C]) or together with Sar1[T39N] ([D] to [F]). LeSec13:GFP distribution ([A] and [D]); SP:RFP distribution ([B] and [E]). Note that SP:RFP is vacuolar in the absence of Sar1[T39N] (B) and is retained in the ER upon Sar1[T39N] expression (E). (C) and (F) are merged images corresponding to (A) and (B) and (D) and (E), respectively.(G) to (J) Effect of Sar1[T39N] in cells coexpressing LeSec13:GFP and GmManI-RFP. LeSec13:GFP signal (G); GmManI:RFP signal (H). Note that GmManI:RFP is redistributed into the ER. (I) and (J) are merged images showing a median (I) and a cortical single optical section (J).Bars = 5 μm.

Effect of BFA on LeSec13:GFP.(A) to (F) LeSec13:GFP cells were transformed by biolistics so as to coexpress GmManI:RFP and were monitored before ([A] to [C]) and after a 20-min treatment with 10 μg·L−1 BFA ([D] to [F]). BFA treatment led to the complete redistribution of GmManI:RFP from the Golgi (A) into the ER (D). Covisualization of LeSec13:GFP (green) and GmManI:RFP (red) at low magnification and high magnification is shown in (B) and (E) and (C) and (F), respectively.(G) to (I) Immunodetection of Sec13 in GmMan1:GFP cells treated for 60 min with BFA. GmMan1:GFP signal (G); anti-AtSec13 immunolabeling (H); corresponding merged image (I).Bars = 5 μm.

Labeling of ER and Golgi in BY-2 Cells Expressing LeSec13:GFP.(A) and (B) Cortical ER in a cell coexpressing AtSec12-YFP (A) and AtBiP:DsRed (B).(C) Merge image for the cell depicted in (A) and (B).(D) An LeSec13:GFP expressing cell bombarded with AtBiP:DsRed; channel selected for green fluorescence.(E) The same cell as in (D), but channel selected for red fluorescence.(F) Merge image for cell depicted in (D) and (E).(G) COPII labeling in a cell expressing LeSec13:GFP, which was bombarded with GmManI-RFP; channel selected for green fluorescence.(H) The same cell as in (G), but channel selected for red fluorescence.(I) Merge image for cell depicted in (G) and (H).(J) and (K) High magnifications of two regions in (I) showing low and high density association of LeSec13:GFP with Golgi stacks.Bars = 5 μm for all panels except (J) and (K) (1 μm).

Analysis of LeSec13:GFP in Relation to the Golgi Apparatus by Time-Lapse Microscopy.LeSec13:GFP-expressing cells were transformed by biolistics to coexpress GmManI:RFP and were monitored using an UltraVIEW RS confocal microscope.(A) and (B) Single optical sections images were acquired approximately every 2.6 s for 182.7 s (see Supplemental Video 2 online). LeSec13:GFP (green signal); GmManI:RFP (red signal).(A) Images taken at 0, 60.9, and 182.7 s.(B) Detailed views of the boxed area visible in Supplemental Video 2 online and in (A). Numbers correspond to the time (in seconds). Arrowheads point to a single Golgi stack during four consecutive frames. Note that at 34.4 s very little LeSec13:GFP is associated with the Golgi stack, whereas at 42.4 s the same Golgi stack is completely surrounded by a halo of LeSec13:GFP.(C) High-magnification pictures of Golgi stacks in face and side view (left and right panels, respectively), showing variable levels of association with LeSec13:GFP.(D) Maximum intensity projection images of a cell coexpressing LeSec13:GFP and GmMan1:GFP viewed at a 18.4-s interval. Arrows point to the same Golgi stack that shows variable levels of association with LeSec13:GFP during time.Bars = 5 μm in (A) and (D) and 1 μm in (B) and (C).

Analysis of Golgi COPII Association in Relation to Golgi Speed.The net velocity of five individual Golgi stacks (from Supplemental Video 2 online) was measured at different times and plotted against the associated LeSec13:GFP fluorescence intensity in a fixed circular 5-μm2 area around each Golgi stack.